A. Field of the Invention
The present invention relates to the formulation of a wet mixture of biochemicals used to enhance stability of biologically active materials, particularly enzymes, both during and after lyophilization. Removal of water from this mixture by lyophilization in the presence of stabilizing agents results in a lyophilized enzyme that is stable at room temperature over extended periods of time. This invention also relates to an improved combination of stabilizing agents useful in a process for lyophilizing an enzyme. The combination is capable of providing enhanced stability of enzymes stored at temperatures above freezing.
B. Background
Enzymes are unstable in aqueous systems at room temperature, and so are typically stored either in a frozen state, or liquid at -20.degree. C. (-70.degree. C. in some cases) in the presence of stabilizers such as glycerol that have low freezing points and low vapor pressures. Even under these storage conditions, repeated thawing and handling at room temperature can lead to loss of activity.
The stabilization of enzymes involves the prevention of irreversible protein denaturation which may occur for a variety of reasons including thermal denaturation and others. Unfolded or denatured proteins have altered structures, which affects the arrangement of the active site and renders them catalytically inactive (Tsou, 1993).
In an attempt to prevent loss of enzymatic activity, a number of stabilization methods have been used. Methods of stabilization may be categorized into four groups: (1) addition of excipients; (2) use of organic solvents (chemical modification); (3) immobilization (conjugation to solid/soluble phases); and (4) protein engineering techniques (Gianfreda and Scarfi, 1991). The method of stabilization described herein involves the use of soluble excipients.
Excipients are inert substances that give a desired characteristic to solvents. Examples of such excipients include sugars, glycerol, polyethylene glycols, amino acids, and other osmolytes. Certain hydrophilic excipients confer stability by making the solvent more polar. An increase in solvent polarity results in an increase in the free energy of transfer for the hydrophobic amino acid moieties from within the protein to the solvent thereby making it more difficult for the protein to unfold (Alonso and Dill, 1991). Glycerol is a common excipient used by enzymologists for storage of enzymes at low temperatures (-20.degree. C.), since some enzymes stored frozen in 50% glycerol buffers may retain much of their initial activity for years.
Combining enzymes of interest with a stabilizing mixture may not alone be sufficient to confer shelf stability. One of the processes used to give proteins long-term stability is drying. When used in conjunction with a stabilizing formulation, drying can yield a very stable product. Methods of drying are generally of two types: (1) air drying and (2) lyophilization. Air drying involves drying under conditions of ambient or elevated temperatures at atmospheric pressure. Lyophilization is a drying process in which water molecules are removed from a frozen solution under a vacuum.
Lyophilization first requires that the aqueous solution be frozen and preferably quick-frozen. One means of quick-freezing a solution is immersing it into liquid nitrogen. A high vacuum is then applied to the frozen sample, which results in sublimation, or vaporization of ice phase at subzero temperatures (primary drying). Residual moisture can be subsequently removed by allowing the temperature to gradually rise (secondary drying) (FTS Systems, Inc., Bulletin #1). The resulting freeze-dried product is a dry, crystalline substance or a powder. Substances that are lyophilized are often hygroscopic, that is, they will tend to absorb atmospheric moisture and lose their stability. In the presence of certain additives, however, it is possible to produce material that is not hygroscopic.
Stabilization and storage of materials using carbohydrates as cryoprotectants has been described by Franks and Hatley (U.S. Pat. No. 5,098,893), but the drying process uses conditions of ambient or elevated temperatures near atmospheric pressure. Use of sugars causes a glassy matrix or rubbery state to form, which when anhydrous, stabilizes the material of interest. Roser, in U.S. Pat. No. 4,891,319 and European Patents WO 87/00196, WO 89/06542, WO 89/06976, EP 0 415 567 A2, describes the process of stabilizing biological materials such as proteins, viruses, and other macromolecules by drying under ambient conditions and at atmospheric pressure in the presence of the carbohydrate trehalose.
The use of sugars such as the disaccharide trehalose as stabilizing agents has been used in conjunction with lyophilization to stabilize polysaccharides (Guthohrlein and Helting, European Patent No. GB 2 009 198 A) and liposomes (Crowe et al., 1987). Lyophilization has also been used to stabilize tumor necrosis factor in the presence of a nonionic surfactant and sugars such as trehalose (Hayashi and Komiya, European Patent No. GB 2 126 588 A). Enzymes that have been stabilized with sugars during lyophilization include phosphofructokinase (Carpenter et al., 1987), which was stabilized with glucose, galactose, maltose, sucrose, and trehalose, and alkaline phosphatase (Ford and Dawson, 1992) which was stabilized with mannitol, lactose, and trehalose.
There is still a need in the art for new and better methods of formulations that stabilize enzymes, particularly enzymes for use in molecular biological applications. The limited availability of freezers and refrigeration in many countries has also created a need to bring enzymes that are stable at ambient temperatures to the user. Thus, it is an object of this invention to provide stabilized enzymes that could be shipped at ambient temperatures and stored for months or years at elevated (or refrigerated [2-8.degree. C.] or ambient) temperatures and still retain all or most of their initial activity levels. It is a further object of this invention to provide certain stabilized enzyme formulations that could be used in various test procedures that are a part of diagnostic kits and other molecular biology research kits. In accordance with these objects, the present invention provides stabilized enzymes which can be stored at temperatures above freezing, and reactivated directly before use.
Therefore, the present invention is directed to providing a formulation that when used with an enzyme or combination of enzymes will not adversely affect enzyme activity prior to and during lyophilization or after reconstitution of the enzyme. The invention is further directed to providing a formulation that when combined with an enzyme or combination of enzymes will not adversely affect enzyme activity prior to and during lyophilization or after reconstitution of the enzyme. This invention is further directed to providing a formulation that when combined with an enzyme or a combination of enzymes will confer greater stability after lyophilization than has heretofore been possible. Yet another aim is to provide a lyophilized enzyme mixture that can be applied to a diagnostic or molecular biology research test.